Particle sorting apparatus and particle sorting method

ABSTRACT

There is provided a particle sorting apparatus, including: an excited light irradiating unit for irradiating an excited light to particles flowing through a flow path; a light irradiating unit for detecting a speed for irradiating a light for detecting a speed to the particles at a position different from the excited light; a light detecting unit for detecting a light emitted from the particles; a calculating unit of an arrival time for individually calculating an arrival time of each particle at a sorting unit being communicating with the flow path from a detection time difference between the light derived from the excited light and the light derived from the light for detecting a speed; and a sorting control unit for controlling sorting of the particles, and a method thereof.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application claims priority to Japanese Priority PatentApplication JP 2013-078212 filed in the Japan Patent Office on Apr. 4,2013, the entire content of which is hereby incorporated by reference.

BACKGROUND

The present disclosure relates to a particle sorting apparatus and aparticle sorting method. More particularly, the present disclosurerelates to a technology for sorting and recovering particles based on aresult of an optical analysis.

SUMMARY

In the related art, to analyze biologically-relevant particles such ascells, microorganisms and liposomes, an optical measuring method using aflow cytometry (flow cytometer) is utilized. The flow cytometer is ananalyzer for irradiating particles flowing through a flow path formedwithin a flow cell or a microchip with light and detecting fluorescenceor a scattered light emitted from each particles.

One of the flow cytometers has a function to sort and recover theparticles having a specific property based on a result of analysis. Inparticular, an apparatus for sorting cells is called as “a cell sorter”.As a sorting method used in the cell sorter, a liquid droplet chargingmethod of charging and separating liquid droplets containing particlesis mainly used (for example, see Japanese Patent Application Laid-openNo. 2009-145213). In an apparatus using the liquid droplet chargingmethod, a fluid discharged from a flow cell or a microchip is liquefied,and liquid droplets are positively (+) or negatively (−) charged,changed their traveling directions by a deflection plate and recoveredby a predetermined container.

However, the sorting method such as the liquid droplet charging methodwhere the liquid droplets are formed is undesirably affected by a changein a measurement environment or a liquid pressure change. A particlesorting apparatus where particles are sorted within a microchip has thenbeen proposed in the related art (see Japanese Patent ApplicationLaid-open No. 2012-127922). The particle sorting apparatus described inJapanese Patent Application Laid-open No. 2012-127922 sucks particles tobe recovered into a negative pressure suction unit within the microchip.Therefore, forming liquid droplets and charging are unnecessary, wherebystable sorting can be done at a high speed without damaging theparticles.

The particle sorting apparatus in the related art is generallycontrolled to acquire the particles to be recovered after apredetermined time from detection at a light detecting unit. The time toacquire the particles from detection is set in advance based on a liquidpressure, a distance from a detection position to a sorting position,etc. However, by the controlling method where an arrival time is fixedin this way, a purity of recovered particles or an acquisition rate isundesirably lowered, once a flow speed of the particles is changed.

On the other hand, the apparatus described in Japanese PatentApplication Laid-open No. 2009-145213 detects a migration speed of eachparticle and controls a timing to charge each particle based on themigrating speed in order to prevent the purity from lowering due to thechange in the flow speed of the particles. In the liquid dropletcharging method, it may only determine that the respective particlesbelong to which liquid droplets. However, in the apparatus for sortingwithin the microchip, fluid mechanism characteristics may be consideredas well as attributes of adjacent particles. Here, the “attributes ofparticles” mean that the particles are to be sorted or not, and the“fluid mechanism characteristics” mean a counter flow generated when apulse signal is risen for acquisition operation.

In the liquid droplet charging method, the liquid droplets arecontrolled. However, in the method for sorting within the microchipdescribed in Japanese Patent Application Laid-open No. 2012-127922, therespective particles should be controlled. In addition, there aredifferences in a path to arrive at an acquisition position and a factoraffecting the arrival of the particles between the liquid dropletcharging method and the method for sorting within the microchip. For theabove reasons, the technology described in Japanese Patent ApplicationLaid-open No. 2009-145213 is not simply applied to the apparatus forsorting within the microchip described in Japanese Patent ApplicationLaid-open No. 2012-127922.

It is desirable to provide a particle sorting apparatus and a particlesorting method for effectively sorting particles within microchip.

According to an embodiment of the present disclosure, there is provideda particle sorting apparatus, including:

-   -   an excited light irradiating unit for irradiating an excited        light to particles flowing through a flow path;    -   a light irradiating unit for detecting a speed for irradiating a        light for detecting a speed to the particles at a position        different from the excited light;    -   a light detecting unit for detecting a light emitted from the        particles;    -   a calculating unit of an arrival time for individually        calculating an arrival time of each particle at a sorting unit        being communicating with the flow path from a detection time        difference between the light derived from the excited light and        the light derived from the light for detecting a speed; and    -   a sorting control unit for controlling sorting of the particles;    -   the flow path and the sorting unit being disposed within a        microchip, and    -   the sorting control unit determining whether or not the        particles are recovered based on data of each particle detected        at the light detecting unit and the arrival time calculated at        the calculating unit of an arrival time.

The sorting control unit calculates an arrival time difference of formerand latter particles, and determines that the particles are notrecovered when the arrival time difference is under a threshold value.

The light for detecting a speed may have a wavelength different from awavelength of the exited light.

In this case, the calculating unit of the arrival time can calculate thearrival time of each particle from the detection time difference betweena scattered light derived from the excited light and a scattered lightderived from the light for detecting a speed.

In addition, the excited light irradiating unit may include two or morelight sources emitting lights having different wavelengths.

The sorting unit may have a negative pressure suction unit beingcommunicated with the flow path.

In this case, the sorting control unit controls an operation of thenegative pressure suction unit based on the data of the respectiveparticles detected at the light detecting unit and the arrival timecalculated at the calculating unit of an arrival time.

Also, the sorting control unit can control a timing to recover theparticles by the sorting unit based on the data of the respectiveparticles detected at the light detecting unit and the arrival timecalculated at the calculating unit of an arrival time.

According to an embodiment of the present disclosure, there is provideda method of sorting particles, including:

-   -   irradiating an excited light to particles flowing through a flow        path disposed within a microchip;    -   irradiating a light for detecting a speed to the particles at a        position different from the excited light;    -   detecting a light emitted from the particles;    -   individually calculating an arrival time of each particle at a        sorting unit disposed within the microchip and being        communicating with the flow path from a detection time        difference between the light derived from the excited light and        the light derived from the light for detecting a speed; and    -   sort-controlling to determine whether or not the particles are        recovered based on data of each particle detected by the light        detection and the arrival time calculated by the arrival time        calculation.

The sorting control may include calculating an arrival time differenceof former and latter particles, and determining that the particles arenot recovered when the arrival time difference is under a thresholdvalue.

The light for detecting a speed having a wavelength different from awavelength of the exited light can be used.

In this case, the calculation of the arrival time may includecalculating the arrival time of each particle from the detection timedifference between a scattered light derived from the excited light anda scattered light derived from the light for detecting a speed.

The irradiation of an excited light can emit different wavelengths fromtwo or more light sources.

Also, the sorting unit may have a negative pressure suction unit beingcommunicated with the flow path. In this case, the sorting controlincludes controlling an operation of the negative pressure suction unitbased on the data of the respective particles detected by the lightdetection and the arrival time calculated by the arrival timecalculation.

In addition, the sorting control can include controlling a timing torecover the particles by the sorting unit based on the data of therespective particles detected by the light detection and the arrivaltime calculated by the arrival time calculation.

According to the present disclosure, the sorting control unit determineswhether or not the particles are recovered based on data of eachparticle detected at the light detecting unit and the arrival timecalculated at the calculating unit of an arrival time. Thus, acquisitionproperties can be improved.

These and other objects, features and advantages of the presentdisclosure will become more apparent in light of the following detaileddescription of best mode embodiments thereof, as illustrated in theaccompanying drawings.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram for schematically showing a configuration of aparticle sorting apparatus according to a first embodiment of thepresent disclosure;

FIG. 2 shows graphs of detection data at a light detecting unit 7 shownin FIG. 1;

FIG. 3 is a block diagram showing a circuit configuration in acalculating unit of an arrival time 8 and a sorting control unit 9 of aparticle sorting apparatus according to an alternative embodiment of thefirst embodiment of the present disclosure;

FIGS. 4A and 4B each shows a processing at an event detection circuitshown in FIG. 3;

FIGS. 5A and 5B each shows a distance at a gating circuit shown in FIG.3;

FIG. 6 shows an operation in an acquisition rate priority mode;

FIGS. 7A and 7B show detection data when the particles are adjacent; and

FIG. 8 shows an operation in a purity priority mode.

DETAILED DESCRIPTION

Hereinafter, an embodiment of the present disclosure will be describedwith reference to the drawings.

The embodiments of the present disclosure will be described in thefollowing order.

-   1. First Embodiment (Example of Particle Sorting Apparatus including    Sorting Control Unit)-   2. Alternative Embodiment of First Embodiment (Example of Particle    Sorting Apparatus including Mode Switching Function)

<1. First Embodiment>

Firstly, a particle sorting apparatus 1 according to a first embodimentof the present disclosure will be explained. FIG. 1 is a diagram forschematically showing a configuration of the particle sorting apparatus1 according to the first embodiment of the present disclosure. FIG. 2shows graphs of detection data at a light detecting unit 7.

[Overall Configuration of Apparatus]

As shown in FIG. 1, the particle sorting apparatus 1 according to thefirst embodiment sorts and recovers particles 10 based on a result of anoptical analysis. The particle sorting apparatus 1 includes a flow path1, a sorting unit 2, an excited light irradiating unit 3, a lightirradiating unit for detecting a speed 4, a light detecting unit 7, acalculating unit of an arrival time 8 and a sorting control unit 9, forexample.

[About Particles 10]

Examples of the particles 10 analyzed and sorted by the particle sortingapparatus 1 according to the first embodiment includebiologically-related particles such as cells, microorganisms andribosomes; and synthesized particles such as latex particles, gelparticles and industrial particles.

Examples of the biologically-related particles include chromosome,ribosome, mitochondoria and organella (cell organelle) that areconstituents of a variety of cells. Examples of the cells include plantcells, animal cells and blood cells. Examples of the microorganismsinclude bacteria such as Bacillus coli, viruses such as a tabacco mosaicvirus, and fungi such as Yeast. Also, the biologically-related particlescan involve biologically-related polymers such as nucleic acids,proteins and composites thereof.

The industrial particles include organic polymer materials, inorganicpolymer materials or metal materials, for example. As the organicpolymer materials, polystyrene, styrene-divinyl benzene, polymethylmethacrylate and the like can be used. As the inorganic materials,glass, silica, magnetic materials and the like can be used. As the metalmaterials, gold colloid, aluminum and the like can be used. In general,shapes of the particles are spherical, but may be non-spherical. Seizesand masses of the particles are not especially limited.

[Flow Path 1]

The flow path 1 is formed within the microchip, into which a liquid (asample liquid) containing the particles 10 to be sorted is fed. Themicrochip including the flow path 1 can be formed of glass or a varietyof plastics (PP, PC, COP, PDSM or the like). Desirably, the material ofthe microchip transmits the light irradiated from the excited lightirradiating unit 3 and the light irradiating unit for detecting a speed4, emits less autofluorescence, has small wavelength dispersion, andinduces less optical errors.

The flow path 1 can be shaped by wet-etching or dry-etching a glasssubstrate, nanoimprinting, injection molding or machining a plasticsubstrate. The microchip can be formed by sealing the substrate in whichthe flow path 1 is shaped with a substrate made of the same material ora different material.

FIG. 1 shows only an area where the flow path 1 is irradiated with theexcited light or the light for detecting a speed. At an upstream sidethereof, a flow path for feeding the sample liquid containing theparticles 10 and a pair of flow paths for feeding a sheath liquid may beprovided. In this case, the flow paths for feeding a sheath liquid meetat both sides of the flow path for feeding the sample liquid. At adownstream side of a meeting point, the flow path 1 is provided. Withinthe flow path 1, the sheath flow surrounds the sample flow, the liquidflows in a state that a laminar flow is formed, and the particles 10 inthe sample liquid flow in an almost row to the flow direction.

[Sorting Unit 2]

The sorting unit 2 sorts the particles 10 to be sorted, and is formedwithin the microchip. The sorting unit 2 is communicated with the flowpath 1 at a downstream side, and is configured of a suction flow path 21and a negative pressure suction unit 22. The configuration of thenegative pressure suction unit 22 is not especially limited as long asmicroparticles to be sorted can be sucked at a predetermined timing. Forexample, the negative pressure suction unit 22 may have theconfiguration that a volume of the negative pressure suction unit 22 canbe expanded by an actuator (not shown) at any timing.

[Excited Light Irradiating Unit 3]

The excited light irradiating unit 3 includes a light source 31 forgenerating an excited light such as a laser light, an optical system 32for shaping a spot shape and a mirror 33. The excited light irradiatingunit 3 irradiates the particles 10 flowing through the flow path 1formed within the microchip with the excited light. In FIG. 1, one lightsource 31 is shown. However, the present disclosure is not limitedthereto, and two or more light sources 31 may be provided. In such acase, the respective light sources 31 may emit lights having differentwavelengths.

[Light Irradiating Unit for Detecting Speed 4]

The light irradiating unit for detecting a speed 4 includes a lightsource 41 for generating a light for detecting a speed, an opticalsystem 42 for shaping a spot shape and a mirror 43. The lightirradiating unit for detecting a speed 4 irradiates the particles 10flowing through the flow path 1 formed within the microchip with thelight for detecting a speed at a position different from theabove-mentioned excited light. The light for detecting a speed may havethe same wavelength as the exited light. From the standpoint ofsimplification of the apparatus configuration, the light for detecting aspeed desirably has the wavelength different from the wavelength of theexited light.

[Light Detecting Unit 7]

The light detecting unit 7 detects a light (a scattered light,fluorescence) generated from the particles 10 flowing through the flowpath 1, and includes a zero-order light removing member 71, mirrors 72 ato 72 d, light detecting units 73 a to 73 d. As the light detectingunits 73 a to 73 d, PMT (Photo Multiplier Tube) or an area imagingsensor such as a CCD or a CMOS element can be used.

In the light detecting unit 7, the light detecting unit 73 a detects aforward-scattered light derived from the excited light, the lightdetecting unit 73 b detects a scattered light derived from the light fordetecting a speed, and the light detecting units 73 c, 73 d detectfluorescence. The light to be detected in the light detecting unit 7 isnot limited thereto, and a side scattered light, a Rayleigh scatteredlight, or a Mie scattered light may be detected. The light detected atthe light detecting unit 7 is converted into an electrical signal.

[Calculating Unit of Arrival Time 8]

The calculating unit of an arrival time 8 individually calculates anarrival time of each particle 10 at the sorting unit 2 beingcommunicated with the flow path from a detection time difference betweenthe light derived from the excited light and the light derived from thelight for detecting a speed. A method of calculating the arrival time isnot especially limited. For example, the arrival time of each particle10 is calculated from the detection time difference between theforward-scattered light (data of Ch1) derived from the excited lightdetected at the light detecting unit 7 and the forward-scattered light(data of Ch2) derived from the light for detecting a speed, as shown inFIG. 2.

Here, the arrival time to the sorting unit 2 can be calculated by asimple linear approximate expression represented by the followingnumerical expression 1. In the numerical expression 1, L1 denotes adistance from a position for irradiating the excited light to a positionfor irradiating the light for detecting a speed, and L2 denotes adistance from the position for irradiating the light for detecting aspeed to the suction flow path 21 of the sorting unit 2 (see FIG. 1). Inthe numerical expression 1, T1 denotes the detection time of the lightderived from the excited light, T2 denotes the detection time of thelight derived from the light for detecting a speed, and (T1−T2) denotesa difference between these detection times (see FIG. 2).(Arrival time)=(L2/L1)×(T1−T2)  [Numerical Expression 1]

A method of calculating the arrival time to the sorting unit 2 is notlimited to the linear approximate expression represented by thenumerical expression 1. Other calculation method such as a polynomialapproximate expression and a look-up table may be used.

[Sorting Control Unit 9]

The sorting control unit 9 controls sorting of the particles 10, anddetermines whether or not the particles 10 are recovered based on thedata of the respective particles 10 detected at the light detecting unit7 and the arrival time calculated at the calculating unit of an arrivaltime 8. The sorting control unit 9 calculates an arrival time differenceof “former” and “latter” particles 10, and determines that the particleshaving the calculated arrival time difference under a threshold valuepreliminary set will “not be recovered”. Thus, when the particles 10flow closely each other, the former particles or the latter particles tobe recovered are prevented from being entrained and acquired wrongly.

In addition, the sorting control unit 9 controls a timing to recover theparticles 10 by the sorting unit 2, for example, by controlling anoperation of the negative pressure suction unit 22, based on theabove-described determination result. In this way, an acquisitionaccuracy of the particles to be intended can be improved to sort theparticles with higher purity or at higher acquisition rate.

[Operation]

Next, an operation of the particle sorting apparatus of this embodimentwill be described. When the particles are sorted by the particle sortingapparatus of this embodiment, the sample liquid containing the particlesto be sorted is fed into a sample inlet disposed within the microchip,and the sheath liquid is fed into a sheath inlet disposed within themicrochip. Then, the particles 10 flowing through the flow path 1 areirradiated with the excited light and the light for detecting a speed ata position different from that of the exited light. In this case, asshown in FIG. 1, the excited light and the light for detecting a speedmay be collected by one light collecting lens 5 to irradiate theparticles 10, but may be collected by different light collecting lenses.

Next, the light detecting unit 7 detects the light emitted from eachparticle 10, and the calculating unit of the arrival time 8 individuallycalculates the arrival time of each particle 10 at the sorting unit 2from the detection time difference between the light derived from theexcited light and the light derived from the light for detecting aspeed. In this case, as shown in FIG. 1, the light derived from theexcited light and the light derived from the light for detecting a speedmay be collected by one light collecting lens 6 to the zero-order lightremoving member 71, but may be collected by different light collectinglenses.

Thereafter, the sorting control unit 9 determines whether or not theparticles 10 are recovered based on the data of the respective particles10 detected at the light detecting unit 7 and the arrival timecalculated at the calculating unit of an arrival time 8. Based on thedetermination result, the sorting control unit 9 controls the timing torecover the particles 10 by the sorting unit 2. For example, when thesorting unit 2 includes the negative pressure suction unit 22 beingcommunicated with the flow path 1, the sorting control unit 9 controlsthe operation of the actuator disposed at the negative pressure suctionunit 22.

As described in detail, the particle sorting apparatus of thisembodiment calculates the arrival time of each particle to the sortingunit, and determines whether or not the particles are recovered in viewnot only of the optical characteristic data of each particle but of thearrival time to the sorting unit. In this way, irrespective of a flowingposition or a flowing status of each particle, the particles can besorted with higher purity or at higher acquisition rate. As a result,acquisition properties can be improved as compared to the particlesorting apparatus in the related art.

As the particle sorting apparatus of this embodiment calculates thearrival time of each particle, a flow rate is less affected by a changein an environment temperature or a residual quantity in a supply tank.Thus, a highly precise control of the flow rate is unnecessary so thatan inexpensive pressure control device can be used and a parts controlof the flow path and an assembly accuracy control can be simplified. Asa result, the manufacturing costs can be decreased.

<2. Alternative Embodiment of First Embodiment>

Next, a particle sorting apparatus according to an alternativeembodiment of the first embodiment of the present disclosure will beexplained. In the particle sorting apparatus according to thealternative embodiment, when the determination whether or not theparticles are recovered is done, a user can select either that the“purity” has priority or that the “acquisition rate” has priority.

FIG. 3 is a block diagram showing a circuit configuration in thecalculating unit of an arrival time 8 and the sorting control unit 9 ofthe particle sorting apparatus according to the alternative embodiment.FIGS. 4A and 4B are diagrams showing a processing at an event detectioncircuit. FIGS. 5A and 5B are diagrams showing a distance at a gatingcircuit. Sorting at a “purity priority mode” or an “acquisition ratepriority mode” can be attained by the circuit configuration shown inFIG. 3, for example.

[Event Detection Circuit]

An event detection circuit reads a waveform of each Ch by applying atrigger with detection signals of Ch1 and Ch2 to calculate a width, aheight and an area shown in FIG. 4A. As to detection data Ch1 concerningthe forward-scattered light derived from the excited light and detectiondata Ch2 concerning the forward-scattered light derived from the lightfor detecting a speed, a time at a waveform center is calculated as thedetection time.

Then, as shown in FIG. 4B, the event detection circuit correlates eachparticle 10 with the detection signals of Ch1 (the forward-scatteredlight derived from the excited light) and Ch2 (the forward-scatteredlight derived from the light for detecting a speed) acquired in timeseries, and packetizes the detection data (events) of the respectiveparticles. A packet includes items that are updated as the subsequentprocessing is proceeded. Flag is basically 1/0, which corresponds toacquisition/non-acquisition determined by each logic. As the detectiontimes, trigger times of Ch1 and Ch2 can be used.

[Calculating Circuit of Arrival Time]

A calculating circuit of an arrival time calculates the arrival timeusing the detection times (T1, T2) of the Ch1 and Ch2 from theabove-described numerical expression 1, which will be a “sorting time”of an event packet.

[Gating Circuit]

A gating circuit determines “acquisition/non-acquisition” of theparticles 10 based on the threshold value preliminary set, and sets“Gate Flag” of an event packet. For example, before starting a gatingacquisition operation, GUI on a computer for controlling is used to plota histogram chart shown in FIG. 5A or a 2D chart shown in FIG. 5B. Agroup of acquired particles (a particle group having intendedproperties) is geometrically bundled and is designated.

A parameter (threshold value) for determining the“acquisition/non-acquisition” may be any of the width, the height andthe area of the detection data acquired in each Ch and a combinationthereof.

[Output Queue Circuit]

An output queue circuit rearranges the detection data (the event) ofeach particle 10 in an arrival order to the sorting unit based on thearrival time to the sorting unit (“Sorting Time”). Thereafter, the“acquisition/non-acquisition” is determined depending on a sorting modeselected by the user such as the “purity priority mode” and the“acquisition rate priority mode”. Based on the result, “Sort Flag” isset.

When the particles 10 flow closely each other, the former particles orthe latter particles may be entrained by one acquisition operation, anda plurality of particles 10 may be recovered twice at the sorting unit2. A method of determining the “acquisition/non-acquisition” isdifferent in the case of the “purity priority mode” or the “acquisitionrate priority mode”, when the particles 10 are adjacent. FIG. 6 shows anoperation in the “acquisition rate priority mode”. FIGS. 7A and 7B showdetection data when the particles are adjacent. FIG. 8 shows anoperation in the purity priority mode.

In the “acquisition rate priority mode”, the number of acquisitionparticles is increased even if the purity of the particles captured isdecreased. As shown in FIG. 6, when the particles 10 flow closely eachother, the particles to be sorted are recovered. In contrast, in the“purity priority mode”, the purity of the particles captured isincreased. When the acquisition particles and the non-acquisitionparticles are adjacent, the acquisition particles are daringly regardedas the “non-acquisition particles” in order to prevent the acquisitionparticles from capturing together with the non-acquisition particles.

In particular, in the “purity priority mode”, as shown in FIG. 8, whenthe detection data (the event) of the particles 10 detected later isadjacent to that of the former particles 10, determination of the“acquisition/non-acquisition” in the former event will be againnecessary. Here, ΔT1 shown in FIG. 7A is a set value and a time toentrain the latter particle (T1=Tn+ΔT1). Also, ΔT2 is a set value and atime to entrain the former particle (T2=Tn+ΔT2).

[Output Timing Generation Circuit]

An output timing generation circuit reads out an event time (Sortingtime) acquired foremost at the output queue, compares it to a ClockCounter value and generates an output timing signal at the time.

[Output Signal Generation Circuit]

An output signal generation circuit detects the output timing signal,and outputs a waveform signal for controlling the actuation device atthe sorting unit 2.

According to the particle sorting apparatus of the alternativeembodiment, when the determination whether or not the particles arerecovered is done, the user can select either that the “purity” haspriority or that the “acquisition rate” has priority. As a result,sorting can be made depending on its purpose. The configurations and theadvantages of the alternative embodiment other than the above aresimilar to those of the above-described first embodiment.

The present disclosure may have the following configurations.

-   -   (1) A particle sorting apparatus, including:    -   an excited light irradiating unit for irradiating an excited        light to particles flowing through a flow path;    -   a light irradiating unit for detecting a speed for irradiating a        light for detecting a speed to the particles at a position        different from the excited light;    -   a light detecting unit for detecting a light emitted from the        particles;    -   a calculating unit of an arrival time for individually        calculating an arrival time of each particle at a sorting unit        being communicating with the flow path from a detection time        difference between the light derived from the excited light and        the light derived from the light for detecting a speed; and    -   a sorting control unit for controlling sorting of the particles;    -   the flow path and the sorting unit being disposed within a        microchip, and    -   the sorting control unit determining whether or not the        particles are recovered based on data of each particle detected        at the light detecting unit and the arrival time calculated at        the calculating unit of an arrival time.    -   (2) The particle sorting apparatus according to (1) above, in        which    -   the sorting control unit calculates an arrival time difference        of former and latter particles, and determines that the        particles are not recovered when the arrival time difference is        under a threshold value.    -   (3) The particle sorting apparatus according to (1) or (2)        above, in which    -   the light for detecting a speed has a wavelength different from        a wavelength of the exited light.    -   (4) The particle sorting apparatus according to (3) above, in        which    -   the calculating unit of the arrival time calculates the arrival        time of each particle from the detection time difference between        a scattered light derived from the excited light and the light        derived from a scattered light for detecting a speed.    -   (5) The particle sorting apparatus according to any of (1)        to (4) above, in which    -   the excited light irradiating unit includes two or more light        sources emitting lights having different wavelengths.    -   (6) The particle sorting apparatus according to any of (1)        to (5) above, in which    -   the sorting unit has a negative pressure suction unit being        communicated with the flow path.    -   (7) The particle sorting apparatus according to (6) above, in        which    -   the sorting control unit controls an operation of the negative        pressure suction unit based on the data of the respective        particles detected at the light detecting unit and the arrival        time calculated at the calculating unit of an arrival time.    -   (8) The particle sorting apparatus according to any of (1)        to (7) above, in which    -   the sorting control unit controls a timing to recover the        particles by the sorting unit based on the data of the        respective particles detected at the light detecting unit and        the arrival time calculated at the calculating unit of an        arrival time.    -   (9) A method of sorting particles, including:    -   irradiating an excited light to particles flowing through a flow        path disposed within a microchip;    -   irradiating a light for detecting a speed to the particles at a        position different from the excited light;    -   detecting a light emitted from the particles;    -   individually calculating an arrival time of each particle at a        sorting unit disposed within the microchip and being        communicating with the flow path from a detection time        difference between the light derived from the excited light and        the light derived from the light for detecting a speed; and    -   sort-controlling to determine whether or not the particles are        recovered based on data of each particle detected by the light        detection and the arrival time calculated by the arrival time        calculation.    -   (10) The method of sorting particles according to (9) above, in        which    -   the sorting control includes calculating an arrival time        difference of former and latter particles, and determining that        the particles are not recovered when the arrival time difference        is under a threshold value.    -   (11) The method of sorting particles according to (9) or (10)        above, in which    -   the light for detecting a speed having a wavelength different        from a wavelength of the exited light is used.    -   (12) The method of sorting particles according to (11) above, in        which    -   the calculation of the arrival time includes calculating the        arrival time of each particle from the detection time difference        between a scattered light derived from the excited light and a        scattered light derived from the light for detecting a speed.    -   (13) The method of sorting particles according to any of (9)        to (12) above, in which    -   the irradiation of an excited light emits different wavelengths        from two or more light sources.    -   (14) The method of sorting particles according to any of (9)        to (13) above, in which    -   the sorting unit has a negative pressure suction unit being        communicated with the flow path, and    -   the sorting control includes controlling an operation of the        negative pressure suction unit based on the data of the        respective particles detected by the light detection and the        arrival time calculated by the arrival time calculation.    -   (15) The method of sorting particles according to any of (9)        to (14) above, in which    -   the sorting control includes controlling a timing to recover the        particles by the sorting unit based on the data of the        respective particles detected by the light detection and the        arrival time calculated by the arrival time calculation.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

The invention is claimed as follows:
 1. A particle sorting apparatus,comprising: an excited light irradiating unit including a first opticalsystem, a first mirror and a first light source for irradiating anexcited light to particles flowing through a flow path; a lightirradiating unit including a second optical system, a second mirror, anda second light source configured to irradiate a light for detecting aspeed of the particles; a light detecting unit for detecting a lightemitted from the particles; a calculating unit for calculating anarrival time of each particle to a sorting unit, the arrival time isassociated with a time difference between a light from the excited lightirradiating unit and a light from the light irradiating unit; and asorting control unit for controlling sorting of the particles; whereinthe flow path and the sorting unit are provided within a microchip,wherein the sorting control unit is configured to determine whether ornot the particles are recovered based on data of the particles detectedat the light detecting unit and the arrival time, and wherein the lightirradiating unit is separate from the excited light irradiating unit. 2.The particle sorting apparatus according to claim 1, wherein the sortingcontrol unit is configured to calculate an arrival time differencebetween former and latter particles, and to determine that the particlesare not recovered when the arrival time difference is under a thresholdvalue.
 3. The particle sorting apparatus according to claim 1, whereinthe light irradiated from the light irradiating unit has a wavelengthdifferent from the exited light irradiated from the excited lightirradiating unit.
 4. The particle sorting apparatus according to claim3, wherein the calculating unit is configured to calculate the arrivaltime of each particle based on the time difference between a scatteredlight derived from the excited light and a scattered light derived fromthe light for detecting a speed.
 5. The particle sorting apparatusaccording to claim 1, wherein the excited light irradiating unitincludes two or more light sources emitting lights having differentwavelengths.
 6. The particle sorting apparatus according to claim 1,wherein the sorting unit has a negative pressure suction unit beingcommunicated with the flow path.
 7. The particle sorting apparatusaccording to claim 6, wherein the sorting control unit is configured tocontrol an operation of the negative pressure suction unit based on thedata of the particles detected at the light detecting unit and thearrival time.
 8. The particle sorting apparatus according to claim 1,wherein the sorting control unit is configured to control a timing torecover the particles by the sorting unit based on the data of theparticles detected at the light detecting unit and the arrival time. 9.A method of sorting particles, comprising: irradiating an excited lightfrom an excited light irradiating unit including a first optical system,a first mirror and a first light source to particles flowing through aflow path provided within a microchip; irradiating a light from a lightirradiating unit including a second optical system, a second mirror, anda second light source to the particles for detecting speed of theparticles; detecting a light emitted from the particles; calculating anarrival time of each particle to a sorting unit based on a timedifference between a light from the excited light irradiating unit and alight from the light irradiating unit; and sort-controlling to determinewhether or not the particles are recovered based on data of eachparticle detected by the light detection and the arrival time, whereinthe light irradiating unit is separate from the excited lightirradiating unit.
 10. The method of sorting particles according to claim9, further comprising calculating an arrival time difference betweenformer and latter particles, and determining that the particles are notrecovered when the arrival time difference is under a threshold value.11. The method of sorting particles according to claim 9, wherein thelight irradiated from the light irradiating unit having a wavelengthdifferent from the exited light irradiated from the excited lightirradiating unit.
 12. The method of sorting particles according to claim11, wherein calculating the arrival time of each particle is based onthe time difference between a scattered light derived from the excitedlight and a scattered light derived from the light for detecting aspeed.
 13. The method of sorting particles according to claim 9, whereinthe excited light includes different wavelengths from two or more lightsources.
 14. The method of sorting particles according to claim 9,wherein the sorting unit has a negative pressure suction unit beingcommunicated with the flow path, and the sort-controlling includescontrolling an operation of the negative pressure suction unit based onthe data of the particles detected by the light detection and thearrival time.
 15. The method of sorting particles according to claim 9,wherein the sort-controlling includes controlling a timing to recoverthe particles by the sorting unit based on the data of the particlesdetected by the light detection and the arrival time calculated by thearrival time calculation.
 16. The particle sorting apparatus accordingto claim 1, wherein the light detecting unit includes a zero-order lightremoving member.
 17. The particle sorting apparatus according to claim1, wherein the arrival time is calculated by expression 1 below:(Arrival time)=(L2/L1)×(T1−T2)  (Expression 1) wherein L1 represents adistance from an irradiation position of the excited light to anirradiation position of the light for detecting a speed, and L2represents a distance from the irradiation position of the light fordetecting a speed to a suction flow path of the sorting unit, T1represents a detection time of the light derived from the excited light,and T2 represents a detection time of a light derived from the light fordetecting a speed.
 18. The particle sorting apparatus according to claim1, wherein the light irradiated from the light irradiating unit has awavelength same as the exited light irradiated from the excited lightirradiating unit.